Cyanohydrins are of importance, for example, for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones and beta-aminoalcohols which are used for obtaining biologically active substances, e.g. pharmaceutical active compounds, vitamins or alternatively pyrethroid compounds.
A cyanohydrin can be prepared by addition of a cyanide group to the carbonyl carbon of an aldehyde or of an unsymmetrical ketone, mixtures of enantiomers of optically active cyanohydrins resulting.
Since in a biologically active mixture of enantiomers only one of the two enantiomers is biologically active, there has been no lack of attempts to find a process for the preparation of the (S)-enantiomer of an optically active cyanohydrin in as high an optical purity as possible.
Thus, in Makromol. Chem. 186, (1985), 1755-62, for example, a process for obtaining (S)-cyanohydrins by reaction of aldehydes with hydrocyanic acid in the presence of benzyloxycarbonyl-(R)-phenylalanine-(R)-histidine methyl ester as a catalyst is described. The optical purity of the (S)-cyanohydrins obtained, however, is highly unsatisfactory.
An enzymatic process for the preparation of optically active (R)- or (S)-cyanohydrins by reaction of aliphatic, aromatic or heteroaromatic aldehydes or ketones with hydrocyanic acid in the presence of (R)-oxynitrilase (EC 4.1.2.10) from Prunus amygdalis or oxynitrilase (EC 4.1.2.11) from Sorghum bicolor is described in EP-A-0 326 063. Examples of the stereo-specific preparation of aliphatic (S)-cyanohydrins are not indicated. This is not surprising, since in Angew. Chemie 102 (1990), No. 4, pp. 423-425 it is stated by inventors who are mentioned in EP-A-0 326 063 that no aliphatic (S)-cyanohydrins can be prepared with hydrocyanic acid from the (S)-oxynitrilase from Sorghum
EP 0 632 130 additionally describes a process in which aliphatic aldehydes or unsymmetrical aliphatic ketones are reacted stereospecifically with hydrocyanic acid and oxynitrilase from Hevea brasiliensis to give (S)-cyanohydrins. The reaction is carried out according to EP 0 632 130, preferably in an aqueous diluent without addition of organic solvents, since these rapidly inhibit the activity of the enzyme, as described in EP 0 632 130.
EP 0 539 767 describes a similar process for the preparation of (S)-cyanohydrins, using specific cyanide group donors instead of hydrocyanic acid. EP 0 539 767 also indicates that organic solvents rapidly inhibit the activity of the enzyme.
The use of recombinant hydroxynitrile lyase from Hevea brasiliensis in an aqueous buffer system is described in Tetrahedron Letters Vol. 52, No. 23, 1996, pp. 7833-7840.
It has now unexpectedly been found that the use of recombinant hydroxynitrile lyase (Hnl) from Hevea brasiliensis makes possible the reaction of a large number of carbonyl compounds, such as, for example, aliphatic, alicyclic, unsaturated, aromatically substituted aliphatic, aromatic, and also heteroaromatic aldehydes and ketones to give the corresponding cyanohydrins, the recombinant Hnl being distinguished by a high resistance to organic solvents.
The invention therefore relates to a process for the preparation of the (S)-enantiomer of an optically active cyanohydrin by reaction of an aldehyde or of a ketone with a cyanide group donor, which comprises reacting the aldehyde or the ketone with a cyanide group donor in an organic diluent in the presence of a recombinant (S)-hydroxynitrile lyase from Hevea brasiliensis and isolating the (S)-cyanohydrin formed from the reaction mixture.
Starting materials employed in the process according to the invention are an aldehyde or a ketone, a cyanide group donor, a recombinant hydroxynitrile lyase and a diluent.
Aldehydes are in this case understood as meaning aliphatic, aromatic or heteroaromatic aldehydes. Aliphatic aldehydes are in this case understood as meaning saturated or unsaturated aliphatic, straight-chain, branched or cyclic aldehydes. Preferred aliphatic aldehydes are straight-chain aldehydes in particular having 2 to 18 C atoms, preferably from 2 to 12, which are saturated or mono- or polyunsaturated. The aldehyde can in this case have both Cxe2x80x94C double bonds and Cxe2x80x94C triple bonds. The aldehyde can be unsubstituted or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups. Examples of aromatic or heteroaromatic aldehydes are benzaldehyde or variously substituted benzaldehydes such as, for example, 3-phenoxybenzaldehyde, additionally furfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde, indole-3-carbaldehyde, naphthalene-l-carbaldehyde, phthalaldehydes, pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde, thiophene-2-carbaldehyde, isophthalaldehyde or pyridine aldehydes etc. Ketones are aliphatic, aromatic or heteroaromatic ketones in which the carbonyl carbon atom is identically or unidentically substituted. Aliphatic ketones are understood as meaning saturated or unsaturated, straight-chain, branched or cyclic ketones. The ketones can be saturated or mono- or polyunsaturated. They can be unsubstituted, or substituted by groups which are inert under reaction conditions, for example by optionally substituted aryl or heteroaryl groups such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, carboxylic acid ester, nitro or azido groups. Examples of aromatic or heteroaromatic ketones are acetophenone, benzophenone etc. Aldehydes and unsymmetrical ketones are preferably reacted.
Aldehydes and ketones which are suitable for the process according to the invention are known or can be prepared in the customary manner.
A possible cyanide group donor is hydrocyanic acid or a cyanohydrin of the general formula R1R2C(OH) (CN). In the formula I, R1 and R2 independently of one another are hydrogen or a hydrocarbon group which is unsubstituted or substituted by groups which are inert under the reaction conditions, or R1 and R2 together are an alkylene group having 4 or 5 C atoms, where R1 and R2 are not simultaneously hydrogen. The hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups. R1 and R2 are preferably alkyl groups having 1 to 6 C atoms, the cyanide group donor is very preferably acetone cyanohydrin.
The cyanide group donor can be prepared according to known processes. Cyanohydrins, in particular acetone cyanohydrin, are also commercially available.
Preferably, hydrocyanic acid or acetone cyanohydrin is employed as the cyanide group donor. The hydroxynitrile lyase employed is recombinant (S)-Hnl from Hevea brasiliensis. Suitable recombinant (S)-Hnl is obtained, for example, from genetically modified microorganisms such as, for example, Pichia pastoris or Saccharomyces cerevisiae. Recombinant (S)-Hnl from Pichia pastoris is preferably employed. By functional overexpression in the methylotrophic yeast Pichia pastoris, this Hnl can be obtained in any desired amount (M. Hasslacher et al., J. Biol. Chem. 1996, 271, 5884). This expression system is particularly suitable for fermentations having a high cell density. Thus, it is possible to obtain approximately 20 g of pure enzyme per liter of fermentation medium. The achievable specific activities of the purified recombinant protein are approximately twice as high as those of the natural enzyme, which was isolated from the leaves of the tree Hevea brasiliensis. After cell disruption, the cytosolic fraction can be used without further purification, by means of which the expenditure of work is minimized. The enzyme is not glycosylated and also has no prosthetic group which would lead to inactivation during removal of the protein moiety. The Hnl can be employed at room temperature for a number of days without significant loss of activity, and is adequately stable at xe2x88x9220xc2x0 C. in the long term. As a result, the possibility results of using the same enzyme batch a number of times. The enzyme is also distinguished by a high resistance to solvents. The possibility therefore exists of employing organic solvents for the enzymatic reaction, which has a favorable effect on the productivity of the respective process.
The hydroxynitrile lyase can be employed in purified or unpurified form, as such or immobilized. The preparation and purification of the hydroxynitrile lyase can be carried out, for example, by precipitation with ammonium sulfate and subsequent gel filtration, for example according to D. Selmar et al., Physiologia Plantarum 75 (1989), 97-101.
The reaction according to the invention is carried out in an organic diluent. Organic diluents which can be used are water-immiscible aliphatic or aromatic hydrocarbons which are optionally halogenated, alcohols, ethers or esters.
Preferably, ethyl acetate, diisopropyl ether, methyl tert-butyl ether and dibutyl ether are used.
The (S)-Hnl can in this case be present either in immobilized form in the organic diluent, but the reaction can also be carried out in a two-phase system, using nonimmobilized (S)-Hnl, the organic diluent employed being a water-immiscible diluent such as, for example, aliphatic or aromatic hydrocarbons which are optionally halogenated, ethers or esters.
Approximately 50 to 300 g of diluent and 200 to 20,000 IU of hydroxynitrile lyase activity, preferably approximately 500 to 5000 IU, are added per g of aldehyde or ketone. An IU (International Unit) in this case expresses the formation of one micromole of product per minute and per gram of enzyme crude isolation. The amount of the respective hydroxynitrile lyase needed is best determined in an activity test, for example according to Selmar et al., Analytical Biochemistry 166 (1987), 208-211.
At least one mole, preferably 1 to 2 mol, of cyanide group donor are added per mole of aldehyde or keto group employed.
The reaction mixture is shaken or stirred at temperatures from approximately 0xc2x0 C. up to the deactivation temperature of the hydroxynitrile lyase, preferably from 20 to 30xc2x0 C. In the course of this, the cyanide group is transferred from the cyanide group donor to the carbonyl carbon atom of the aldehyde or ketone employed and the (S)-enantiomer of the optically active cyanohydrin corresponding to the aldehyde or ketone employed is mainly formed. The progress of the reaction can in this case be monitored, inter alia, by gas chromatography.
After reaction has taken place, the cyanohydrin formed can be extracted from the reaction mixture with the aid of an organic solvent which is not miscible with water, for example aliphatic or aromatic optionally halogenated hydrocarbons, e.g. pentane, hexane, benzene, toluene, methylene chloride, chloroform, chlorobenzenes, ethers such as, for example, diethyl ether, diisopropyl ether or esters, for example ethyl acetate or mixtures of such solvents. Should the purity of the extracted product not be adequate, a purification operation can follow. The purification can be carried out by a known method and takes place best chromatographically.